Project Summary The glomerular basement membrane (GBM) is a major component of the glomerular filtration barrier. Of the nine major GBM proteins, mutations in at least 4 of them cause human disease. Pierson syndrome (congenital nephrotic syndrome with ocular and nervous system abnormalities) is caused by laminin beta2 (LAMB2) null mutations; in contrast, LAMB2 missense mutations cause congenital nephrotic syndrome with less severe and highly variable extrarenal manifestations. On the other hand, Alport syndrome is caused by mutations affecting any one of three collagen IV genes (COL4A3, A4, and A5). These diseases have very different presentations and rates of progression to ESRD, but the fact that a GBM defect is the initiating insult in both demonstrates the importance of investigating GBM structure and function in order to better understand how to treat patients. For over 20 years we have been interested in understanding the makeup of the glomerular filtration barrier and how it becomes damaged and leaky to plasma proteins using our mouse models of Pierson and Alport syndromes. Having determined why certain missense LAMB2 mutations cause nephrotic syndrome, here we now propose to test protein therapy approaches designed to remedy defects in the GBM using both transgenic and intravenous protein therapy modalities. Our preliminary data show that full-sized laminin trimers injected i.v. reach the GBM, become stably integrated into the GBM, and moderately improve the filtration barrier in Lamb2 null mice. This proof of concept suggests that improving GBM structure via the bloodstream is a viable therapeutic option. We will use rationally designed chimeric matrix proteins that are much smaller than full- sized laminin trimers to attempt to improve laminin polymerization in the GBM of novel mutant mice with laminin polymerization defects, in the context of both nephrotic syndrome and Alport syndrome. In addition, state of the art gene expression profiling of single cells will be used to determine how proper laminin polymerization impacts podocyte homeostasis, as well as that of other glomerular cells. The results of these studies will provide important new insights into laminin and basement membrane biology and lead to potential therapies for human glomerular disease involving GBM defects.